This revised proposal is in response to the Program Announcement PA-12-060 entitled 'Solicitation of Validated Hits for the Discovery of in vivo Chemical Probes' (R01). The goal of this proposal is to synthesize and select novel chemical scaffolds as antagonists of APOBEC3G (A3G) binding to inhibitory RNA. These will serve as molecular probes to determine the role that RNA binding to A3G has in preventing host defense against an HIV infection. A3G is effective in host defense against HIV if it can evade destruction by the HIV protein known as Vif (made during late infection) and if it becomes packaged with viral particles and thereby enters cells with virions. The bulk of A3G preexisting in cells prior t an infection is largely inert as its activity is severely attenuated through its nonselective formation of ribonucleoprotein complexes with cellular RNAs. RNA binds to the N-terminus of A3G and allosterically inhibits the ssDNA binding and deaminase domain in the C-terminus. High throughput screening by OyaGen, Inc has identified three validated chemical scaffolds for their ability to: (1) antagonize RNA binding to A3G and (2) preemptively activate A3G in cells to the extent that incoming HIV replication is inhibited. In the proposed research OyaGen, Inc and Sanford/Burnham will collaborate to: (1) apply rational design to synthesize chemical modifications of three validated A3G- selective antiviral compounds, (2) select those with enhanced activity as antagonists of A3G:RNA binding and antiviral activity, (3) select the ultimate probe(s) based on enhanced activation of A3G DNA mutagenic activity and selectivity for cellular A3G-RNA complexes, and. (4) triage compounds to select those with drug-like characteristics based on in vitro ADME/T and in vivo PK in mice. These probes will usher in a significant paradigm change as they will enable studies and monitoring of the mechanism of action of APOBEC3G as it naturally is expressed in human cells during a live virus infection and, in the future, in animal models.